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Cold Spring Harbor protocols Cold Spring Harb Protoc 2009 Aug;2009(8):pdb.prot5275 Dissection, plating, and maintenance of dorsal roo Albuquerque C;Joseph D;Choudhury P;MacDermott A 101524530 20147251 1940-3402 (Print) Any format Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY : Copyright Compliance Guidelines bramelma N/A $12.00 Oberst, Rachel - TN: 246404 Other CAHSLA,GMRRG,OutLibGMR 1.513.558-0173 1.513.558-4899 hsldd@ucmail.uc.edu Ariel: ariel2.uc.edu Ariel,Email(PDF),Mail GMR-RL-RECIPROCAL***Prefer Odyssey if possible*** Received Jul 15, 2011 08:14 by NYURPM as Refer to Resource Libraries 1559-6095 (Electronic) Verify: PubMed

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Cite as: Cold Spring Harb Protoc; 2009; doi:10.1101/pdb.prot5275

Protocol
Dissection, Plating, and Maintenance of Dorsal Root Ganglion Neurons for Monoculture and for Coculture with Dorsal Horn Neurons
Cristvo Albuquerque, Donald J. Joseph, Papiya Choudhury, and Amy B. MacDermott1
Department of Physiology and Cellular Biophysics and Department of Neuroscience, Columbia University, New York, NY 10032, USA
1Corresponding

author (abm1@columbia.edu )

INTRODUCTION
Dorsal root ganglion (DRG) neurons grow readily alone or in coculture with embryonic dorsal horn (DH) neurons and even appear to enhance survival of the DH cells. Furthermore, DRG neurons grow long neurites in the presence of nerve growth factor, forming many glutamatergic synapses onto DH neurons, if present. Otherwise (i.e., in monocultures), no synapses can be detected electrophysiologically. When preparing cocultures, use DRGs already removed from the same embryos used to prepare DH neurons, but after dissecting the dorsal horns; the DRGs are less sensitive to delay in dissection and dissociation, as long as they are left in culture medium. Microisland and mass cultures require different proportions of DRG and dorsal horn neurons. Synaptic interactions in the microisland culture of DRG and DH neurons are more intense than mass culture because the growing, branching DRG axons are confined to a limited area, and thus make many synapses onto each target DH neuron.

RELATED INFORMATION
For information on preparing DH neurons for coculture, see Dissection, Plating, and Maintenance of Dorsal Horn Neuron Cultures (Albuquerque et al. 2009a). For protocols describing other techniques used in the preparation of neuronal cultures, see Preparation of Coverslips for Neuronal Cultures (Albuquerque et al. 2009b), and Dissection, Plating, and Maintenance of Cortical Astrocyte Cultures (Albuquerque et al. 2009c).

MATERIALS
Reagents All solutions must be sterile. Astrocytes cultured on coverslips in 35-mm dishes Prepare astrocytes as described in Dissection, Plating, and Maintenance of Cortical Astrocyte Cultures (Albuquerque et al. 2009c). Only use dishes where the astrocytes are confluent and translucent, as assessed by inverted phase-contrast microscopy. Carbon dioxide (CO2)

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Ethanol (70% [in a squirt bottle] and 95%) Leibovitzs L-15 medium (ice-cold) (Invitrogen 11415064) MEM + B-27 Modified Eagle medium (S-MEM; Invitrogen 11380037) Nerve growth factor (NGF; 10 g/mL; Roche Applied Science 11362348001) Filter through a 0.22-m filter before use. Rat (pregnant, 16th day of gestation) Trypsin (2.5%; Invitrogen 15090046) U/FDU Equipment Beaker (100-mL) Centrifuge Dishes (culture, plastic, 35- and 60-mm) (BD Falcon 351008 and 351007, respectively,) Forceps (coarse and two pairs of fine) Fume hood Hemacytometer Ice Mammalian cell culture incubator preset to 37C (water-jacketed, equilibrated with 5% CO2) Paper towels Pasteur pipettes Flame-polish the tips of the pipettes with a Bunsen burner before use. Scissors (iridectomy, small) Scissors (large and small) Tubes (capped centrifuge, sterile, 15-mL (BD Falcon 352096) Tubes (capped centrifuge, sterile, 50-mL (Corning 430828) (optional; see Step 9)

METHOD
Preparation

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1. Before beginning dissection, place three 60-mm culture dishes containing cold L-15 medium on ice. Place a 35-mm dish containing 2 mL of S-MEM in the incubator. 2. Remove all of the IMDM for astrocytes from the 35-mm dishes containing the cultured astrocytes. Replace with 1.5 mL MEM + B-27. 3. Clean the dissection area. Wipe with 70% ethanol. 4. Cover the bottom of a 100-mL beaker with paper towels. Fill the beaker with 95% ethanol. Place the dissection instruments in the beaker. Lining the beaker with paper towels protects the tips of the fine forceps. 5. Anesthetize a pregnant rat in the 16th day of gestation with CO2 until loss of righting reflex and pinch response occurs. Perform the anesthesia under a fume hood. 6. Use large scissors to kill the rat by thoracotomy. Embryo Removal 7. Wipe the abdomen of the rat with 70% ethanol. Cut through the skin. Separate it from the peritoneum. Retract the skin flaps from the surgical area. 8. Squirt the exposed peritoneum with 70% ethanol. Cut the peritoneum. Use coarse forceps to suspend the uterine tube, being careful to avoid the external surfaces of the rat. 9. Cut a section of the uterine tube containing two to three embryos (seen as bulges in the uterus). If transport is required, transfer the cut section of uterine tube to a 50-mL centrifuge tube containing 35 mL of ice-cold L-15 medium. 10. Place the cut piece of uterine tube containing the embryos in a 60-mm dish containing ice-cold L-15 medium. 11. Cut open the uterine tube. Remove two embryos. Only two embryos are required; reserve the third embryo as a back-up if needed. 12. Use forceps to transfer the embryos by their head (to avoid damaging the spinal cord) to a fresh 60-mm dish containing ice-cold L-15 medium. Spinal Cord Removal 13. Cut off the head, umbilical cord, and tail from each embryo. 14. Using fine forceps and small iridectomy scissors, place the embryos on their backs. 15. Pin an embryo by its shoulders with the forceps. 16. Using small iridectomy scissors, cut down the ventral length of the embryo.

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17. With the embryo still pinned down, remove the viscera with another pair of forceps. Take care not to puncture the spinal cord during removal of the unwanted tissue. The ventral side of the vertebral column should now be visible (see Fig. 1A ).
Figure 1. Photographs and schematics of spinal cord dissection. All photographs are of tissues in 35-mm dishes. (A) Ventral view of an embryo with viscera removed and vertebral column exposed. The head has been removed. (B) Spinal cord with attached DRG following its removal from the vertebral column. A schematic representation is shown below the photograph. (C) Spinal cord with DRGs plucked off. A schematic representation is shown below the photograph. (D) Flattened spinal cord with two dorsal horns partially cut away. A schematic representation is shown below the photograph. The dotted lines indicate the approximate site of cutting. View larger version (41K): [in this window] [in a new window]

18. Insert the lower blade of small iridectomy scissors through the neck opening into the vertebral cavity. By alternately cutting at either side of the midline, make two parallel cuts all the way down to the tail stub. The cuts should be made as lateral as possible to the midline. 19. Use fine forceps to lift the "strip" of bone. Take care not to damage the underlying spinal cord. 20. Insert the tips of two pairs of fine forceps in the space between a side of the spinal cord and the lateral aspect of the vertebral column. Gently pull the forceps apart, pulling the spinal cord away from the vertebrae. Repeat this process, gently but quickly, down the length of the cord on each side. Do not pull out the spinal cord. This step loosens the DRGs from the vertebrae and considerably improves DRG recovery. 21. Hold the carcass down with one pair of forceps. Use a second pair of fine forceps to hold the spinal cord by its most rostral aspect (being sure to take hold of some of the outer meninges). Slowly pull the cord away from the vertebral cavity. The cord should come out with most of the DRGs still attached (see Fig. 1B). 22. Transfer each spinal cord with attached DRGs to a 35-mm culture dish containing 2 mL of prewarmed SMEM (from Step 1). DRG Extraction and Plating 23. Hold the spinal cord down with one pair of fine forceps. With a second pair of forceps, pluck the DRGs from the cord (like grapes), holding them by roots. Discard the spinal cord. Alternatively, if preparing DH neurons for coculture, leave the DRGs in the medium at this point and process the spinal cord as described in Dissection, Plating, and Maintenance of Dorsal Horn Neuron Cultures (Albuquerque et al. 2009a). 24. Add 200 L of trypsin to the dish containing the DRGs. Incubate for 20-25 min at 37C. 25. Add 2 mL of MEM + B-27 to the dish. 26. Taking care to collect most of the DRGs, transfer the medium to a 15-mL tube. Use a fire-polished Pasteur pipette to dissociate the tissue by repeatedly aspirating and expelling the tissue.

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Cold Spring Harb Protoc -- Albuquerque et al. 2009 (8): pdb.prot5275 Figure 1

Page 1 of 1

Click on image to view larger version.

Figure 1. Photographs and schematics of spinal cord dissection. All photographs are of tissues in 35-mm dishes. (A) Ventral view of an embryo with viscera removed and vertebral column exposed. The head has been removed. (B) Spinal cord with attached DRG following its removal from the vertebral column. A schematic representation is shown below the photograph. (C) Spinal cord with DRGs plucked off. A schematic representation is shown below the photograph. (D) Flattened spinal cord with two dorsal horns partially cut away. A schematic representation is shown below the photograph. The dotted lines indicate the approximate site of cutting.

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27. Centrifuge the sample at 1300 rpm for 8 min. 28. Remove the supernatant. Resuspend the pellet in 2 mL of MEM + B-27. Neuron Plating and Maintenance 29. Count the neurons using a hemacytometer. 30. Dilute the dissociated cells to the appropriate concentration with MEM + B-27. For cocultures with DH cells, double the indicated concentrations and mix 1:1 with 2X DH neuron suspensions before plating; the final number of cells/plate should be as indicated in Step 31, in a final volume of 2 mL of medium. i. For mass culture, dilute to 6.25 x 104 cells/mL. ii. For microisland cultures, dilute to 5.0 x 103 cells/mL. 31. Plate 2 mL of medium containing the dispersed DRG cells (i.e., 1.25 x 105 cells for mass culture, 1.0 x 104 cells for microislands) into 35-mm dishes containing astrocyte-coated coverslips (from Step 2). 32. Add 10 L of NGF to each dish. 33. Forty-eight hours after plating, add 10 L of U/FDU. 34. Once per week, feed the cells by replacing 1 mL of the MEM + B-27 medium with fresh medium (without U/FDU). Add 10 L of fresh NGF at each feeding. To reduce the possibility of cross-contamination, frequently change the Pasteur pipette used to aspirate the old medium. Figure 2 shows a modified phase-contrast micrograph of a single microisland with confluent astrocytes, a DH and a DRG neuron.
Figure 2. Differential interference contrast image of a microisland with astrocytes, a DH and a DRG neuron.

View larger version (54K): [in this window] [in a new window]

DISCUSSION

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Cold Spring Harb Protoc -- Albuquerque et al. 2009 (8): pdb.prot5275 Figure 2

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Click on image to view larger version.

Figure 2. Differential interference contrast image of a microisland with astrocytes, a DH and a DRG neuron.

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Primary neuronal cultures have been used extensively to characterize the functional properties of synapses and to study the synaptic effects of pharmacological agents, proteins, and genetic modification. Coculture preparations comprised of dissociated DRG and DH neurons have been used as a model system for synaptic transmission at primary afferent synapses. Additionally, this model permits simultaneous measurement of post-synaptic response from multiple cell populations in response to DRG activation.

REFERENCES
1. Albuquerque C, Joseph DJ, Choudhury P, MacDermott AB. 2009a. Dissection, plating, and maintenance of dorsal horn neuron cultures. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5274. [Abstract/Free Full Text] 2. Albuquerque C, Joseph DJ, Choudhury P, MacDermott AB. 2009b. Preparation of coverslips for neuronal cultures. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5272.[Abstract/Free Full Text] 3. Albuquerque C, Joseph DJ, Choudhury P, MacDermott AB. 2009c. Dissection, plating, and maintenance of cortical astrocyte cultures. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5273. [Abstract/Free Full Text]

Recipe
MEM + B-27

Reagent

Amount to add

B-27 supplement, serum-free (50X; Invitrogen 17504044) D-(+)-glucose, SigmaUltra (200 mg/mL; Sigma G7528) MEM vitamin solution (100X; Invitrogen 11120052) Minimum essential medium (MEM; Invitrogen 11090081) Penicillin-streptomycin (Invitrogen 15140122) (optional) Filter through a 0.22-m filter.

4 mL 8 mL 2 mL 185 mL 1 mL

Recipe
U/FDU
5-fluoro-2'-deoxyuridine (1 mM; Sigma F0503) Uridine (1 mM; Sigma U3750)

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Filter through a 0.22-m filter before use.

Copyright 2009 by Cold Spring Harbor Laboratory Press. Terms of Service. All rights reserved. Anyone using the procedures outlined in these protocols does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in these protocols and has no liability in connection with their use. All materials used in these protocols, but not limited to those highlighted with the Warning icon, may be considered hazardous and should be used with caution. For a full listing of cautions, click here. All rights reserved. No part of these pages, either text or images, may be used for any reason other than personal use. Reproduction, modification, storage in a retrieval system or retransmission, in any form or by any means-electronic, mechanical, or otherwise-for reasons other than personal use is strictly prohibited without prior written permission.

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C. Albuquerque, D. J. Joseph, P. Choudhury, and A. B. MacDermott Preparation of Coverslips for Neuronal Cultures Cold Spring Harb Protoc, August 1, 2009; 2009(8): 10.1101/pdb.prot5272. [Abstract] [Full Text]

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